APC/C Dysfunction Limits Excessive Cancer Chromosomal Instability
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Published OnlineFirst January 9, 2017; DOI: 10.1158/2159-8290.CD-16-0645 RESEARCH ARTICLE APC/C Dysfunction Limits Excessive Cancer Chromosomal Instability Laurent Sansregret1, James O. Patterson1, Sally Dewhurst1, Carlos López-García1, André Koch2, Nicholas McGranahan1,3, William Chong Hang Chao1, David J. Barry1, Andrew Rowan1, Rachael Instrell1, Stuart Horswell1, Michael Way1, Michael Howell1, Martin R. Singleton1, René H. Medema2, Paul Nurse1, Mark Petronczki1,4, and Charles Swanton1,3 Downloaded from cancerdiscovery.aacrjournals.org on September 26, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst January 9, 2017; DOI: 10.1158/2159-8290.CD-16-0645 ABSTRACT Intercellular heterogeneity, exacerbated by chromosomal instability (CIN), fosters tumor heterogeneity and drug resistance. However, extreme CIN correlates with improved cancer outcome, suggesting that karyotypic diversity required to adapt to selection pressures might be balanced in tumors against the risk of excessive instability. Here, we used a functional genomics screen, genome editing, and pharmacologic approaches to identify CIN-survival factors in diploid cells. We find partial anaphase-promoting complex/cyclosome (APC/C) dysfunction lengthens mitosis, suppresses pharmacologically induced chromosome segregation errors, and reduces naturally occurring lagging chromosomes in cancer cell lines or following tetraploidization. APC/C impairment caused adaptation to MPS1 inhibitors, revealing a likely resistance mechanism to therapies targeting the spindle assembly checkpoint. Finally, CRISPR-mediated introduction of cancer somatic mutations in the APC/C subunit cancer driver gene CDC27 reduces chromosome segregation errors, whereas reversal of an APC/C subu- nit nonsense mutation increases CIN. Subtle variations in mitotic duration, determined by APC/C activity, influence the extent of CIN, allowing cancer cells to dynamically optimize fitness during tumor evolution. SIGNIFICANCE: We report a mechanism whereby cancers balance the evolutionary advantages associ- ated with CIN against the fitness costs caused by excessive genome instability, providing insight into the consequence of CDC27 APC/C subunit driver mutations in cancer. Lengthening of mitosis through APC/C modulation may be a common mechanism of resistance to cancer therapeutics that increase chromosome segregation errors. Cancer Discov; 7(2); 1–16. ©2017 AACR. INTRODUCTION based on the degree of CIN has revealed that extremes of CIN are associated with improved prognosis, lending credence to Chromosome missegregation leads to abrupt changes in the “just-right” threshold of genomic instability sufficient gene expression and protein stoichiometry that result in a for tumor adaptation proposed by Cahill and colleagues strong negative selection pressure when occurring in most (22–26). Excessive CIN appears deleterious for cell fitness, diploid cell types, but which are tolerated in aneuploid cancer and, accordingly, enhancing chromosome missegregation has cells (reviewed in ref. 1). At least part of the selection against been proposed as an approach to target CIN cancer cells aneuploidy relies on p53, which limits cell propagation after (18, 27, 28). Hence, selection could favor the attenuation of chromosome missegregation and genome-doubling (2–4). CIN in human cancer to prevent excessive genome instability Under selective pressure, however, chromosomal instabil- while ensuring sufficient karyotypic instability to foster adap- ity (CIN) enables cells to explore various karyotypic states, tation to a changing environment. Here, we explore cellular allowing the eventual emergence of subclones with improved mechanisms contributing to the adaptation of excessive CIN fitness, a recurrent mode of adaptation observed in fun- in human cancer. gal pathogens, yeast, and mammalian cells, and a cause of treatment failure (5–17). Murine models largely support the notion that CIN favors tumor formation, but conversely RESULTS excessive CIN appears to suppress tumorigenesis, analogous Experimental Model for CIN Threshold to mutational meltdown and error-prone catastrophe in bac- and Tolerance terial and viral genetics (18–21). Although CIN has been gen- erally associated with poor prognosis, patient stratification To investigate how cells respond and adapt to whole- chromosome missegregation, we sought a method to induce CIN in diploid cells that was amenable to high-throughput screening. Given the crucial role of the spindle assembly 1The Francis Crick Institute, London, United Kingdom. 2The Netherlands checkpoint (SAC) for chromosome segregation fidelity, we 3 Cancer Institute, Amsterdam, the Netherlands. CRUK UCL/Manchester Lung took advantage of reversine, an inhibitor of the SAC kinase Cancer Centre of Excellence. 4Boehringer Ingelheim, Vienna, Austria. MPS1 encoded by the gene TTK (29, 30). We investigated Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). whether reversine titration could tune the frequency of seg- regation errors in cells. The hTERT-immortalized diploid J.O. Patterson and S. Dewhurst contributed equally to this article. epithelial cell line RPE1 and near-diploid HCT116 colorec- Corresponding Authors: Charles Swanton, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, United Kingdom. Phone: 00 44 20 tal cancer cells were chosen for their karyotypic stability 3796 2047; E-mail: [email protected]; and Mark Petronczki, and refractoriness to CIN, which is largely due to their Boehringer Ingelheim, NTC Discovery, Dr. Boehringer Gasse 5-11, Vienna, functional p53 pathway (2). We measured segregation error A-1121, Austria. E-mail: [email protected] rates by centromeric FISH from RPE1 daughter cell pairs doi: 10.1158/2159-8290.CD-16-0645 born during acute reversine exposure in mitosis. Reversine ©2017 American Association for Cancer Research. increased the error rate per chromosome pair per division in a FEBRUARY 2017 CANCER DISCOVERY | OF2 Downloaded from cancerdiscovery.aacrjournals.org on September 26, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst January 9, 2017; DOI: 10.1158/2159-8290.CD-16-0645 RESEARCH ARTICLE Sansregret et al. dose-dependent manner, from 0.00027 in DMSO-treated tested and subunits targeted suggest that our results are a cells (approximately 1 error per 165 divisions, assuming consequence of on-target reduction in APC/C activity. a diploid karyotype with equal error rates for all chromo- The SAC functions by inhibiting the APC/C during the somes) to 0.021 in 250 nmol/L (1 error per 2 divisions), 0.055 early stage of mitosis until chromosome bi-orientation is in 500 nmol/L (1.3 chromosome errors per division on aver- achieved at metaphase, which in turn silences SAC signaling age), 0.183 in 750 nmol/L (4.2 chromosomes per division), and triggers anaphase onset by allowing APC/C bound to and 0.232 in 1 μmol/L reversine (5.3 chromosomes per div- its coactivator CDC20 to degrade securin and cyclin B (29). ision; Fig. 1A; Supplementary Table S1). Reversine treatment The duration from nuclear envelope breakdown to anaphase resulted in the p53-dependent expression of p21 (CDKN1A) (NEBD–anaphase) therefore represents the time during which and reduced proliferation in RPE1 and HCT116 cells, reflect- the SAC is active and monitors chromosome attachment to ing the activation of a cellular stress response to aneuploidy the mitotic spindle (32). Of note, complete pharmacologic following a single passage through mitosis (Supplementary inhibition of the APC/C or genetic ablation of the co-activator Fig. S1 A–S1F; ref. 2). TP53 RNAi increased the proliferation CDC20 robustly arrests cells at metaphase (33, 34), indicating of RPE1 cells in reversine, reflecting greater tolerance to CIN that the RNAi conditions used here result in partial APC/C and aneuploidy (Supplementary Fig. S1B; ref. 2). Phospho- loss of function. APC/C subunit RNAi rescued cell-cycle arrest rylation of p53 at serine 15 was not detectable after reversine following MAD2 RNAi or reversine-induced SAC impairment, treatment, and the ATM inhibitor KU-55933 did not block and reduced the number of micronuclei, suggesting that p21 induction in reversine, suggesting that an ATM-medi- fewer segregation errors were occurring, in contrast to TP53 ated DNA-damage response was not the underlying cause siRNA which did not reduce micronuclei formation (Fig. 1E; for cell-cycle arrest (Supplementary Fig. S1G and S1H). We Supplementary Fig. S3A–S3C). FISH analyses confirmed that conclude that reversine titration allows control over the rate CDC16 RNAi or transient inhibition of proteasomal degrada- of chromosome segregation errors in otherwise diploid cells, tion with MG132 efficiently reduced the rate of segregation thereby mimicking varying levels of CIN induction. errors in reversine-treated cells (Fig. 1F; Supplementary Table S1; We next used reversine titration to examine the selective Supplementary Fig. S3D–S3F). These results are consistent advantage of a genetic background permissive to CIN in with previous observations that imposing a long metaphase response to increasing levels of CIN, by comparing HCT116 arrest (>80 minutes) using 12 μmol/L of the APC/C inhibitor wild-type and TP53−/− isogenic cell lines (31). TP53 disruption proTAME can rescue segregation errors in MAD2-depleted provided a clear proliferative advantage in low concentrations cells (35).